Human cripto protein

ABSTRACT

A new human gene designated as &#34;CRIPTO&#34; gene has been identified and cloned. CRIPTO gene products and derivatives thereof have been obtained and various utilities of the same have been described. Association of CRIPTO gene with cancers, such as colorectal cancer and breast carcinoma, has been indicated.

BACKGROUND OF THE INVENTION

The present invention is related generally to the isolation and cloningof genes and obtaining products encoded by the gene. More particularly,the present invention is related to the isolation, cloning, sequencingand expression of the human CRIPTO gene and producing an isolated,substantially pure gene products including mRNA and recombinant CRIPTOprotein.

"CRIPTO" is a new human gene which has never been previously described.The gene has been isolated, cloned and completely sequenced. FIG. 1shows the nucleotide sequence of the CRIPTO cDNA and the amino acidsequence deduced therefrom. FIG. 1 also shows the amino acid sequence ofthe natural CRIPTO protein and FIG. 2 the amino acid sequence of therecombinant E. coli derived CRIPTO protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (which consists of FIGS. 1A through 1C) shows the nucleotidesequence of the human CRIPTO gene cDNA and the corresponding amino acidsequence.

FIG. 2 shows the amino acid sequence of the human CRIPTO protein as itis recombinantly reproduced in E. coli.

FIG. 3 is a comparison of the amino acid sequence of the human CRIPTOgene to several prior art proteins.

FIG. 4 demonstrates the focus forming activity of the human CRIPTO genewhen transfected into NIH 3T3 cells.

FIG. 5 is a Northern blot showing expression of the CRIPTO gene byvarious human colon tumor cell lines.

FIG. 6 is a Northern blot demonstrating that the CRIPTO gene is notexpressed in normal human colon tissue.

DETAILED DESCRIPTION Isolation and Characterization of Human CRIPTO cDNA

In screening 3×10⁷ independent clones of a human teratocarcinoma NT2D1cell line cDNA library that was expressed in λgt10 and that wasoriginally derived from NT2D1 poly(A)+RNA to isolate a full-lengthglucose-6-phosphate dehydrogenase (G6Pd) cDNA, 16 different clones wereidentified (Persico et al., Nucleic Acid Research, 14:2511-2522, 1986).One of these clones exceeded the expected size for the G6PD mRNA.Restriction mapping and sequencing showed that the aberrant cDNA whichwas approximately 5 kb in length to be a composite of two separatecoding entities. A nucleotide segment of 2.8 kb corresponded to G6PDwhile the remaining 2.2 kb fragment (16B6 cDNA) had no relationship tothe G6PD gene. The 16B6 cDNA fragment was used to probe the same NT2D1cDNA library to isolate a full-length cDNA.

From several positive clones, 10 clones were isolated and subcloned intopUC18 after EcoR1 digestion. Analysis by restriction enzyme mapping andagarose gel electrophoresis demonstrated that the size of the variouscDNA inserts varied from about 0.9 Kb to 2.0 Kb. The two largest cDNAclones, p3B2 and p1C1, and the shortest p2B3, were sequenced by theSanger method. The complete nucleotide sequence has been deposited inthe EMBL Gene Data Bank. The open reading frame of 564 base pairs codesfor a protein of 188 amino acids in length (FIG. 1). Proteolyticcleavage sites are present in this protein designated CRIPTO at V-A(amino acid residues 28-29 and 159-160), R-K (residues 111-112), K-K(residues 126-127) and R-T-T-T (residues 171-174). One potentialasparagine glycosylation sequence (Asn-Arg-Thr) is present at residues79-81.

Production and Purification of Recombinant CRIPTO Protein in E. Coli

The buffers are prepared as follows:

Buffer A: 25% sucrose, 10 mM Tris-HCl (pH 8.0), ImM EDTA, 150 mM NaCland 10 μg lysozyme.

Buffer B: 10 mM Tris-HCl (pH 7.6), ImM EDTA and 0.5% Triton X-100.

Buffer C: 0.1% SDS. 0.05M Tris-HCl (pH 8.0), 0.ImM EDTA, 5 mM DTT and0.20M NaCl.

1. Grow an inoculum of suitably transformed strain of bacteria in LBbroth containing 100 μg/ml of ampicillin overnight at about 32° C.

2. Dilute the bacterial culture 100-fold in LB broth and grow at 32° C.until OD₆₀₀ reaches 0.2.

3. Shift the bacterial culture to 44° C. for 20 minutes and then to 42°C. for 4 hours until OD₆₀₀ reaches 1.7.

4. Spin 50 ml of the bacterial culture at 5,000 g for 10 minutes at 4°C. and resuspend the bacterial pellet in 10 ml LB broth at roomtemperature (RT).

5. To 10 ml of frozen buffer A add 10 ml of bacterial suspension anddefrost at RT prior to incubation for 15 minutes at 37° C.

6. Spin at 27,000 g for 10 minutes at 4° C.

7. Resuspend the pellet in 3 ml of buffer B and spin at 15000 rpm for 15minutes at 4° C.

8. Repeat step #7 three times, saving the supernatant each time.

9. Sonicate the final spheroplast suspension 6 times for 30 seconds at40 watts.

10. Divide into 4 Eppendorff tubes and spin at RT in a microfuge at12,000 g for 10 minutes.

11. Discard the supernatant and resuspend the pellet of inclusion bodiesin 1 ml of IM urea. Incubate for 30 minutes at 37° C. Spin 10 minutes inmicrofuge at 12,000 g.

12. Repeat step #11 twice.

13. Each pellet of the inclusion bodies is then dissolved in 200 μl ofLaemmli sample buffer and analyzed by SDS-PAGE. Alternatively, resuspendinclusion body pellets in 600 μl of buffer C to solubilize therecombinant CRIPTO protein.

Recombinant CRIPTO Protein Characterization

1. The CRIPTO cDNA is used to produce a recombinant CRIPTO protein in E.coli as described above. The amino acid sequence of the CRIPTO proteinis shown in FIG. 2. The inclusion body pellets are resuspended in 600 μlof buffer C and incubated at 37° C. for 18 hours to achieve almost 100%solubilization of the CRIPTO protein.

2. A partial solubilization is achieved in either 0.1M Tris-HCl buffer(pH 8.0) containing 6M guanidine HCl, 10M reduced glutathione and IMoxidized glutathione or in 0.05M Tris-HCl buffer (pH 8.0) containing ImMEDTA, 0.1M NaCl, 8M urea diluted with nine volumes of 0.05M KH₂ PO₄ (pH10.7), 1 mM EDTA (pH 8.0) and 0.05M NaCl.

3. Following solubilization and SDS-PAGE analysis, the CRIPTO proteinhas a Mr of about 20,000 to 22,000.

A deposit of the cloned cDNA of the CRIPTO gene has been made at theATCC on Feb. 28, 1990 under accession number 61412. The deposit shall beviably maintained, replacing if it becomes non-viable during the life ofthe patent, for a period of 30 years from the date of the deposit, orfor 5 years from the last date of request for a sample of the deposit,whichever is longer, and upon issuance of the patent made available tothe public without restriction in accordance with the provisions of thelaw. The Commissioner of Patents and Trademarks, upon request, shallhave access to the deposit.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

The term "substantially pure" as used herein means as pure as can beobtained by standard isolation and purification techniquesconventionally known to one of ordinary skill in the art.

The term "a reactive amount" as used herein means a quantity of theprotein that would function in a manner desired in a particularapplication or utility of the protein.

As mentioned above, CRIPTO is transcribed into 2200 nucleotide long mRNAwhich is translated into a protein of 188 amino acid residues. Table 1shows the expression of CRIPTO gene in humans and mice. The gene isactive in teratocarcinoma cells, but inactive both in normal and othertransformed cells and shut off when the teratocarcinoma cells areinduced to differentiate by retinoic acid.

The amino acid sequence of CRIPTO protein was screened against arepresentative protein sequence database (Microgenie, Beckman). Thissearch revealed that the CRIPTO protein is similar to several proteins,some of which are shown in FIG. 3. The similarity is restricted to a.sup.˜ 40 amino acid long, cysteine-rich, sequence known as the EGF-likesegment. Besides the six cysteine residues in the characteristic spatialarray, other amino acids are conserved among these proteins, e.g. theglycine, phenylalanine and tyrosine residues boxed in FIG. 1.

Transforming potential of the CRIPTO gene

It has been shown that certain oncogenes, such as K-FGF, c-sis,proto-dbl and c-erbB-2 can transform murine fibroblasts when theirexpression is driven by a strong promoter. Similarly, TGFα and EGF genesunder the control of a strong promoter can induce transformation andtumorigenicity in fibroblasts.

To investigate whether the human CRIPTO gene has these properties, itscDNA was introduced into an expression vector in which transcription iscontrolled by the RSV long terminal repeats (LTR) (Gorman et al, 1982).The construct was transfected into NIH3T3 cells and its focus-formingactivity was monitored (FIG. 4). In this experiment, the CRIPTO cDNAinduced foci of transformed cells at an efficiency of 600 focus-formingunits per pmol of DNA.

When CRIPTO cDNA was placed into a retroviral expression vector plasmidand transfected into mouse NIH-3T3 fibroblasts and into mouse NOG-8mammary epithelial cells, in both of these cell types overexpression ofthis gene resulted in the in vitro transformation of these cells.(Tables 2 and 3).

In addition, substantially pure, isolated, recombinant CRIPTO protein(rCRIPTO) was obtained from a baculo virus expression vector in whichthe CRIPTO cDNA had been integrated. The availability of the CRIPTO cDNAand rCRIPTO protein now make it possible to detect cells or tissuesexpressing the CRIPTO gene. Various utilities of the CRIPTO cDNA andCRIPTO protein are now described.

Application and utilities of the CRIPTO cDNA and CRIPTO protein

Since the mRNA for the CRIPTO gene is expressed in approximately 60% to70% of human colon tumor cell lines and at an equal frequency in primaryhuman colon tumors, but not in normal human colon tissue (see Northernblot FIGS. 5 and 6), therefore, expression of CRIPTO mRNA and CRIPTOprotein in a tissue would be a major tumor specific marker for thediagnosis and eventual prognosis of different types of cancer such ascolorectal cancer. In addition, the CRIPTO gene maps to human chromosome3 potentially at a region where deletions frequently occur and wheresuch deletions have been found to be associated with a subset of primaryhuman breast tumors and with a majority of small cell lung carcinomas.Hence, a loss of heterozygosity for this gene and/or a loss of or areduction in CRIPTO mRNA expression due to deletions of one or bothalleles of the CRIPTO gene may serve as adjunct tumor specific markersfor other types of human cancer. Additionally, it has beenexperimentally demonstrated that introduction and subsequentoverexpression of the human CRIPTO gene in a retroviral expressionvector can lead to the in vitro transformation as detected byfocus-forming activity or by anchorage-independent growth in soft agarof mouse NIH-3T3 fibroblast cells and of mouse NOG-8 mammary epithelialcells (Tables 2 and 3) indicating a role of this gene in the neoplasticprocess. Based on these facts, the availability of the CRIPTO cDNA andrecombinant CRIPTO protein now allows the following applications:

1. The molecularly cloned, full-length human CRIPTO cDNA can benick-translated, isotopically labeled, for example, with ³² Pnucleotides and subsequently used as a probe for the analysis ofSouthern blots containing endonuclease digested DNA preparations toascertain if there are amplifications, rearrangements, deletions orrestriction fragment length polymorphisms of the CRIPTO gene in normalversus tumor tissue.

2. The labeled nick-translated CRIPTO cDNA can also be utilized for theanalysis of Northern blots that contain poly(A)+RNA to determine therelative levels of CRIPTO mRNA expression in various normal andpathologic tissue samples.

3. The CRIPTO cDNA can be cloned into an SP6/T7 pGEM expression vectorand the like and can then be used to generate a corresponding cRNAantisense riboprobe. This antisense riboprobe could then be labeled with³⁵ S nucleotides and utilized as a suitable probe for in situ RNA:RNAhybridization for histologic localization in normal or pathologic cellsexpressing CRIPTO mRNA.

4. CRIPTO sense oligonucleotides can be chemically synthesized and canbe used as appropriate probes in a polymerase chain reaction (PCR) forpotential detection of low levels of CRIPTO mRNA and for amplificationof CRIPTO genomic sequences for subsequent isolation and cloning.

5. The CRIPTO cDNA can be utilized to generate either expression vectorplasmids for transfection or to generate replication defectiverecombinant ecotropic or amphotropic retroviral expression vectors forinfection into cells for determining whether overexpression of this genein vitro might lead to malignant transformation or might alter thegrowth or differentiation properties of different mammalian cell types.

6. The CRIPTO cDNA when placed in an appropriate expression vectorplasmid or in a comparable retroviral expression vector in the oppositeorientation can be used to generate antisense mRNA. Such antisenseexpression vectors can then be used to transfect or to infect normal andmalignant cells in vitro in order to determine whether endogenous CRIPTOexpression is important in maintaining the proliferation,differentiation or transformation of these cells.

7. Nonderivatized or thio-derivatized CRIPTO antisense oligonucleotidescan be chemically synthesized and used to treat cells in vitro similarlyas described in #6 above. Additionally, antisense CRIPTOoligonucleotides can be incorporated into liposomes for site-directeddelivery in vivo to tumors when appropriate tumor-specific monoclonalantibodies are also incorporated into these same vesicles.

8. The CRIPTO cDNA can be placed into various bacterial, yeast, insectbaculo virus or mammalian expression vectors in order to obtainsufficient quantities of a potentially biologically active, recombinantCRIPTO protein.

9. A recombinant CRIPTO protein can be used to generate a panel ofpolyclonal (in rabbits, sheep, goat or pigs) and mouse monoclonalantibodies such that these immunological reagents can be used to screenfor CRIPTO protein expression in normal and pathologic human and animaltissue samples by immunocytochemistry, by Western blot analysis, byenzyme-linked immune substrate assay (ELISA), by radioimmunoassay (RIA)and the like.

10. Since the CRIPTO protein is a member of the epidermal growth factor(EGF) supergene family that contains a variety of peptide mitogens andgrowth inhibitors, a biologically active recombinant CRIPTO protein canbe used to determine if this peptide has any growth regulatory activityon a variety of normal and tumor cells in vitro.

11. Additionally, a recombinant CRIPTO protein can be iodinated and canbe utilized to identify and characterize specific cell surface receptorsfor this potential growth modulatory peptide using conventional chemicalcross-linking techniques.

It is noted that the methodologies for the above noted utilities arewell known to one of ordinary skill in the art and no novel techniquesare seen involved in making such usages. A composition of matter, inaccordance with the present invention, comprises a reactive amount ofthe rCRIPTO protein in a sterile, non-toxic carrier or vehicle.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

                  TABLE 1    ______________________________________    EXPRESSION OF CRIPTO GENE IN HUMANS AND MICE                         Total Poly (A).sup.+                         RNA   RNA    ______________________________________    Organs and tissues    Placenta (human)       --      --    Testis (mouse)         --      --    Cell lines    HL60 (undifferentiated human myeloid                           --      ND    cells)    JEG (human choriocarcinoma cells)                           --      --    PA-1 (human neuroblastoma cells)                           --      ND    Ca-Ma (human mammary carcinoma cells)                           --      ND    Human T lymphocyte     --      ND    HeLa                   --      --    NA43 (human fibroblasts)                           ND      --    NT2D1 (undifferentiated human teratocar-                           +       +    cinoma cells)    ΔNT2D1 (differentiated human teratocar-                           --      --    cinoma cells)    Term placenta fibroblasts                           ND      --    Term placenta primary culture                           ND      --    F9 (undifferentiated mouse teratocarcinoma                           +       ND    cells)    ΔF9 (differentiated mouse teratocarcinoma                           --      --    cells)    NIH3T3 (mouse fibroblasts)                           --      ND    ______________________________________     ND, Not determined

                  TABLE 2    ______________________________________    Anchorage-Independent Growth of Mouse NOG-8 Mammary    Epithelial Cells Transfected with a Human cripto cDNA in a    RSV Expression Vector Plasmid    Clone             Total number of colonies/dish    ______________________________________    NOG-8 (parental nontransfected)                       10 ± 5.sup.a (-)    2E                  1690 ± 80 (+++)    2L                 925 ± 70 (++)    2F                175 ± 25 (+)    2H                166 ± 10 (+)    ______________________________________     .sup.a 2 × 10.sup.4 cells were seeded in 0.3% soft agar over a 0.8%     agar overlay in 35 mm tissue culture dishes. Cultures were maintained for     14 days prior to staining of the cells with nitroblue tetrazolium.     Colonies greater than 50 μm were scored and counted on an Artek colony     counter. Results are the average from four separate dishes ± S.D.     Numbers in parenthesis represent relative amounts of cripto mRNA as     detected in cells following Northern blot hybridization with a labeled     human cripto cDNA insert.

                  TABLE 3    ______________________________________    Focus-Forming Activity of Human cripto cDNA in a RSV    Expression Vector Plasmid after Transfection into Mouse    NIH-3T3 Cells    Clone              Total number of foci/dish    ______________________________________    NIH-3T3 (parental nontransfected)                       .sup.  5 ± 2.sup.a    Clone γ9     82 ± 5    ______________________________________     .sup.a 2 × 10.sup.3 cells were seeded in 35 mm dishes and maintaine     for 2 weeks prior to staining with crystal violet.

What is claimed is:
 1. An isolated, substantially pure, recombinantCRIPTO protein having the amino acid sequence shown in FIG.
 2. 2. Acomposition of matter, comprising the rCRIPTO protein of claim 1 in asterile, nontoxic carrier.
 3. An isolated, substantially pure,recombinant CRIPTO protein encoded by a nucleotide sequence shown inFIG.
 1. 4. The recombinant CRIPTO protein of claim 3 wherein the proteinis encoded by a full length human CRIPTO gene.